Activated Natural Killer Cells Suppress Myelopoiesis in Mice with Severe Combined Immunodeficiency

The in vivo effect of natural killer (NK) cell activation on aulologous myelopoiesis was studied in an environment deficient of functional Tand B cells. Administration of 3.6-bis[2-(Dimethylamino)-ethoxy]-9H-xanthen-9-one dihydrochloride) Tilorone) or recombinant interleukin-2 (rIL-2) to mice with severe combined immunodeficiency (C. B. -I7 scid/scid) resulted in an increase in YAC-1 lysis by their splenocytes as well as bone marrow cells. Recombinant IL-2 furthermore led to a fivefold increase in the cellularity of the spleen. When assayed against human NK/lymphokine-activated killer (LAK) target, K562 cell line. the IL-2-activated mouse cells exhibited no cytotoxicity across the species barrier. Both agents induced a profound suppression of myelopoietic progenitor cells as measured in a 7-day granulocyte-macrophage colony forming cell (GM-CFC) assay. We conclude that the presence of neither functional T nor B cells is necessary for NK cells to mediate inhibition cf myelopoiesis in the autologous host.     

In Vivo Generation of Mouse Natural Killer Cells: Role of the Spleen and Thymus

The role of the spleen and thymus was investigated in the natural killer (NK) cell system. These NK cells have the ability to kill a variety of tumour cells as demonstrated in vitro in short-term ⁵¹Cr release assays. The work presented deals with three observations: (1) the effect of splenectomy on the levels of NK activity in the blood and lymph nodes. (2) the effect of splenectomy on the reconstitution of irradiated animals with bone marrow cells, and (3) the level of NK activity in adult thymectomized, irradiated animals which were reconstituted with bone marrow or thymus cells from either high or low NK activity animals. Thus, for the third point, chimaeras were established between histocompatible strains of mice A. BY (a low NK strain), and C57B1/6 (a high NK strain). The ability of T cells from one strain could then be observed to either help or suppress the NK activity of the bone-marrow-derived cells. The data presented show that the absence of a spleen does not affect MK activity or reconstitution of NK cells in irradiated animals. Further, T cells from one strain do not affect NK activity of animals reconstituted with bone marrow cells from a histocompatible strain. Thus T cells from a low NK stain (A. BY) did not suppress the high activity of C57B1/6 cells, and, conversely, the C57B1/6 T cells did not compensate for the low NK activity of A. BY cells.     

Overnight incubation of mouse spleen cells in recombinant IL-2 generates cytotoxic cells with NK characteristics from precursors enriched with or devoid of LGL.

Interleukin-2 (IL-2) augments natural killer (NK) activity as well as generating effector cells named lymphokine activated killer cells (LAK) which are capable of lysing a wide spectrum of target cells. A large body of evidence has been accumulated to evaluate the relationship between NK and LAK cells and conflicting results have been reported. Our study was addressed to further analyse this relationship and in particular to investigate whether in a short incubation IL-2 is merely capable of augmenting the activity of pre-existing killer cells, or whether it can also promote the differentiation of precursor cells. Eighteen-hour culture of mouse spleen cells in human recombinant IL-2 induced a DNA-synthesis-independent generation of cytotoxic cells bearing an NK phenotype (aGM-1+, Thy1.2+/-, CD8-, CD4-). These were generated from precursor cells also bearing an NK phenotype, recovered either from low density Percoll fractions enriched in lytic cells with LGL morphology as well as from high density fractions devoid of LGL and cytotoxic activity.     

Recombinant interleukin 2 allows the differentiation of Thy 1.2+ LAK cells from nude mouse spleen cells

Culture of nude mouse spleen cells with recombinant human interleukin 2 (r-IL 2) resulted in the proliferation and generation of lymphokine-activated killer (LAK) cells which could lyse a variety of tumor cells. Flow cytometry study indicated that nude mouse spleen cells contained almost no Thy 1.2+ cells at the initial times of the culture, whereas LAK cells obtained from nude mouse spleen cells by culture with r-IL 2 (nude-LAK cells) expressed high intensity of Thy 1.2 antigen and lymphokine-activated cell-associated (LAA) antigen. The cytotoxic activity of nude-LAK cells was greatly reduced by treatment with anti-Thy 1.2 antibody plus complement but not with anti-Ly 1.2 or Ly 2.2 antibody plus complement treatment. Moreover, nude-LAK cells were resistant to the treatment with anti-asialo GM1 antibody plus complement, in contrast to resident nude natural killer (NK) cells. These data strongly suggested that r-IL 2 allowed the nude mouse spleen cells to differentiate into Thy 1.2+, Ly 1-,2-, asialo GM1- LAK cells which were distinct from Thy 1.2+, Ly 2+, asialo GM1- LAK cells induced from normal mouse spleen cells.     

Cell Proliferation during the Maturation of Natural Killer Cells

The requirement for cell division during the maturation of natural killer (NK) cells was studied by following the appearance of donor-type NK cells in irradiated mice injecied with bone marrow cells and by blocking the cell division at different times during this development. Irradiation (700 rad) or treatment with hydroxyurea (1 mg/g body weight, twice daily) of the recipieni mice 7 days after the bone marrow cell inoculation inhibited the appearance of normal NK cell levels, suggesting that the NK cell progenitors are dividing cells. Blocking of the cell division in chimeras that had already developed high NK levels decreased the splenic NK activity, indicating the presence of a dividing NK cell population at this stage of maturation. These results are in accordance with the concept that mature NK cells are nondividing cells but are derived from actively proliferating progenitors in the bone marrow, and some of the first NK cells appearing in the spleen from the bone marrow can still be dividing.     

Natural Killer Cells and Cytotoxic T Cells Induce DNA Fragmentation in Both Human and Murine Target Cells in Vitro

DNA fragmentation induced by cytolytic lymphocytes in human erythromyeloid cell line K562 and murine T lymphoma cell line YAC-1 was investigated by means of agarose gel electrophoresis. Murine natural killer (NK) and cytotoxic T (Tc) cells induced DNA fragmentation in YAC-1 cells, with the fragments being approximately multiples of 180 bp. More significantly, murine NK cells can induce a similar pattern of DNA fragmentation in human K562 cells. Therefore, cytolytic lymphocytes can induce apoptosis or programmed cell death in human target cells.     

Analysis of low natural killer cell activity in 89Sr-treated mice

Treatment of mice with the long-lived bone-seeking radioisotope 89Sr results in the selective irradiation and destruction of the bone marrow. This is accompanied by a marked reduction in natural killer cell activity against YAC-1 lymphoma [NK(YAC-1)]. To test for the presence of cellular suppressors of NK(YAC-1) in 89Sr-treated mice, in vitro and in vivo cell mixture protocols were used. In vitro, we did not observe any specific inhibitory effect of spleen cells from 89Sr-treated mice on NK(YAC-1) activity of normal spleen cells. The NK(YAC-1) activity of 89Sr-treated mice, measured in vivo by their ability to clear radiolabeled YAC-1 cells from the lungs, was impaired. However, spleen cells from 89Sr-treated mice, when adoptively transferred with normal spleen cells, failed to inhibit the NK(YAC-1) activity of the latter in the lung clearance assay. Further, when normal spleen cells were injected into 89Sr-treated mice, the ability of the transferred cells to mediate in vivo activity was not suppressed in the 89Sr-treated host. These experiments support the suggestion that the low NK(YAC-1) activity in 89Sr-treated mice is not mediated by suppressor cells, but may be due to the destruction of the marrow microenvironment which is essential for the generation of functional NK(YAC-1) cells.     

Syngeneic pregnancy induces overexpression of natural killer T cells in maternal liver

Conditions such as stress, infection, autoimmune disease, etc. elevate the number and function of extrathymic T cells that are generated mainly in the liver. As primitive, self-reactive clones of T cells that coexpress receptors of the natural killer (NK) lineage, they mediate cytotoxicity against altered self, malignant and infected cells and have the unique potential to rapidly secrete large amount of T helper 1 (Th1) or Th2 cytokines. To elucidate whether some of these changes occur even during the syngeneic pregnancy, we made phenotypic and functional characterization of mononuclear lymphatic cells (MNLCs) isolated from the liver and spleen of pregnant C57BL/6 mice, testing their cytotoxicity against syngeneic thymocytes as well as against NK- and lymphokine-activated killer (LAK)-sensitive targets. The data have shown that on the sixteenth day of syngeneic pregnancy TCRint, NK1.1+ and IL-2Rbeta+ cells were accumulated in the liver, while the quantities of CD4+ and CD8+ T cells and total number classical NK (NK1.1+CD3- or IL-2Rbeta+CD3-) cells were increased in the spleen. Pregnancy-activated hepatic and splenic MNLCs were more cytotoxic against syngeneic thymocytes, YAC-1 and P815 targets, suggesting that the maternal liver is a main producer of autoreactive NKT clones, which subsequently augment NK- and LAK cell-mediated cytotoxicity in the liver and spleen.     

Immunomodulatory Activity of A Novel Nucleoside, 7-thia-8-oxoguanosine: I. Activation of Natural Killer Cells in Mice

We reported recently that a novel immunomodulator, 7-thia-8-oxoguanosine (7T80G)² inhibited formation of pulmonary melanoma metastases (1), prevented against viral infection in mice (2) and potentiated the efficacy of a weakly immunogenic leukemia vaccine (3). Since certain tumor metastases and virus infected cells are targets to natural killer cells (NK cells), we now investigated whether 7T80G is capable of activating NK cells in mice using NK cell sensitive YAC-1 and B16 and NK cell insensitive P815 targets. CBA/CaJ spleen cells incubated in vitro with 7T80G at concentrations ranging from 0.005 to 0.5 mM responded with increased NK cell activity (32-62 %) compared to controls (4-8%) to YAC-1 targets. Similar levels of augmentation in NK cell activity were observed when 40-168 mg/kg of 7T80G was administered in vivo. In addition to the spleen, 7T80G activated NK cells in the bone marrow (BM), the lungs, the liver, and in peritoneal exudate cells (PE). Although 7T80G elicited activation of NK cells was observed as early as three hours after treatment, the maximal activity was observed after 24 h in the spleen; 12 h in the BM; 48 h in the lungs, and 72 h in PE. Administration of the drug by s.c, i.v., and i.p. routes all induced activition of NK cells in spleen, BM and PE. 7T80G was found to activate NK cells in seven inbred and an outbred mouse strain, suggesting that the induced cytotoxicity against allogeneic and syngeneic tumor cells is not strain specific as well as independent of MHC restriction. C3H/He, CBA/CaJ and BDF/1 displayed higher levels of increased NK cell activity, whereas AKR mice were low responders. Low concentrations of IL-2 (0.25-5 U/ml) that induce little or no NK cell activity, when used in combination with 7T80G, elicited significant enhancement of NK cell cytotoxicity. In contrast, IFN and 7T80G showed no such synergism.     

Development of Natural Killer Cells: Comparison of the Maturation Rates in Different Lymphoid Compartments

By means of semisyngeneic bone marrow transplantation, the appearance of donor-type natural killer (NK) cells in different organs (spleen, blood, lungs) of the recipients was studied. Seven days after the transplantation the first donor-type NK cells appeared in all these organs, and adult NK levels were reached simultaneously within a couple of days. The first NK cells to appear in every organ divided rapidly (sensitive to hydroxyurea) and contained a high proportion of Thy-1+ cells. These data suggest that NK cell precursors mature locally and simultaneously in different organs.     

Gelatin sponge model of effector recruitment: tumoricidal activity of adherent and non-adherent lymphokine-activated killer cells after culture in interleukin-2

This study examined the specific tumoricidal activity of lymphokine-activated killer (LAK) cells derived from tumor-infiltrating lymphocytes that prevent the growth of secondary tumors in animals harboring progressing primary tumors. A pre-implanted gelatin sponge was employed to capture infiltrating host effectors during the expression of concomitant tumor immunity. Additionally, this study compared the cytolytic activity of these sponge-derived cells with those of counterpart splenic lymphocytes. The cells from both sources were cultured for 4 days in IL-2 to generate LAK cells which were further expanded in IL-2-containing medium for up to 11 days. The cytotoxic activities of these cells were measured in a Chromium-51 release assay. The data revealed that the culture of splenic, or sponge-derived lymphocytes results in the emergence of non-adherent and adherent cell populations with LAK activity. The 4-day sponge-derived LAK cells (adherent and non-adherent) exhibited significant cytolysis of EMT6 cells while the spleen-derived counterparts showed minimal cytotoxicity toward these targets. Some NK activity in LAK cells derived from both sources was evident by their lysis of YAC-1 cells. LAK cells from both sources were incapable of lysing histo-compatible EL-4 (H-2b) tumor cells. The lysis of the EMT6 cells by the sponge-derived LAK cells was maintained over an 11-day period of culture in IL-2. Conversely, the spleen-derived LAK cells were unable to significantly lyse EMT6 cells during this period of in vitro culture. These results show the superior specific tumoricidal activity of LAK cells derived from lymphocytes mediating tumor rejection in vivo (sponge-derived) over that of counterpart splenic lymphocytes.     

rIL-4 differentially regulates rIL-2-induced murine NK and LAK killing in CD8+ and CD8- precursor cell subsets

Interleukin 4 (IL-4) and IL-2 have complementary or synergistic roles in many aspects of lymphocyte development. IL-2 supports the induction of cytolytic activity in cytotoxic T lymphocyte (CTL), natural killer (NK), and lymphokine-activated killer (LAK) cells. IL-4 has also been shown to support CTL and LAK in primary murine spleen cell culture. This report demonstrates that IL-4 selectively down-regulates IL-2 inducible murine CD8- precursors of NK cells. For maximal regulatory effect it is necessary to add IL-4 to cultures before 40 h. Enrichment for NK1.1+ cells failed to recover precursor cells which are down-regulated in overnight cultures or can be cultivated in vitro to yield NK cytolytic activity. Furthermore, phenotypic analysis of effector cells demonstrated a marked inhibition of development of NK1.1+ cells in cultures containing IL-4 plus IL-2 versus IL-2 alone. Thus, it appears that IL-4 down-regulates the precursors of murine NK cells by inhibiting proliferation and/or development. In addition, we show that IL-2-induced murine LAK activity mediated by CD8- precursor cells is unaffected by IL-4, while CD8(+)-derived LAK cells are up-regulated by co-culture with IL-4 and IL-2. Analysis of these data relative to reports documenting down-regulation of human LAK by IL-4 suggests that in vitro cultured, IL-2-activated murine NK cells are the correlates to what are commonly described as human LAK cells. The discrepancy may stem from differences in the characteristics of target cells used in the murine versus the human systems. These results clarify the conflicting reports on the effect of IL-4 on killing activity.     

Differentiation of Macrophage Precursors to Cells with LAK Activity under the Influence of CSF-1 and High Dose IL-2

Mouse macrophage precursor cells with natural killer (NK) like activity, derived from in vitro culture of light-fraction-bone marrow cells in the presence of colony-stimulating factor 1 (CSF-1) and low dose IL-2, were incubated with high dose (1000 U/ml) IL-2. After 3 days of incubation, cells had developed from NK like (killing Yac-1 but not P815) into LAK-like (killing both YAC-1 and P815) effector cells. Morphological studies revealed that LAK activity occurred at the time when macrophage precursors with NK like activity containing few cytoplasmic granules had further differentiated into cells with abundant azurophilic granules in their cytoplasma. Proliferation of macrophage-precursor derived NK/LAK-like cells was dependent on the presence of colony-stimulating factor, generation of cytoplasmic granules was induced by IL-2 in a dose dependent way. Flow cytometric analysis showed that macrophage precursor-derived LAK effectors were positive for NK 1.1 but almost negative for F4/80. When the same starting cell population was cultured in the presence of 200 U/ml Interferon gamma (IFN gamma), proliferation was completely stopped and within 3-4 days all cells differentiated into mature macrophages expressing F4/80. In context with our previous data, we describe here a continuum of development from agranular macrophage precursors to granular cells with NK-like activity and further to cells with LAK activity under the influence of CSF-1 as growth factor and IL-2 as granule- and cytotoxicity-inducing factor.     

Altered Natural Killer and Natural Cytotoxic Cellular Activities in lpr Mice

Three strains of mice bearing the autosomal recessive lpr gene (MRL, C57BL/6, and C3H) that had spontaneously developed a lupus-like disease were studied sequentially for functional natural killer (NK) and natural cytotoxic (NC) cell activity. Natural killing was impaired in spleen and bone marrow cells from all the lpr strains, as well as from the congenic strain MRL--+/+, which develops a late onset lupus-like disease. The NK cell activity was found to be depleted as early as 2 months of age in all lpr strains, and decreased further with age. NK activity was augmentable by Poly I:C and interleukin 2 (IL-2), suggesting that the residual cells can respond to NK modulators. In contrast with NK cell activity, NC activity was not decreased in lpr mice but could be augmented by IL-3-rich supernatants. The spontaneous decrease in NK cell activity was associated with an increased autologous plaque-forming cell (APFC) response to bromelin-treated mouse red blood cells, which is produced primarily by B cells possessing the Ly-1 phenotype (Lyt-1+ B). When NK cell activity was increased by exogenous administration of Poly I:C, the APFC response diminished. Treatment of spleen cells with anti-asialo GM1 prior to Poly I:C treatment resulted in a decreased NK response but increased both APFC and Lyt-1+ B cells. The possible regulation of autoreactivity by NK cells is discussed.     

Enumeration andCharacterization of Human Killer and Natural Killer Cells by a Modified Single-Cell Assay

Human natural killer (NK) and killer (K) cells were assayed in a modified single-cell cytotoxicity assay using poly-L-lysine-coated cover slips. When human Chang liver cells were used as targets, 20% of the lymphocytes formed conjugates and 2% were active NK cells. When anti-Chang antibodies were present, the proportion of target-binding cells (TBC) increased to 30% and that of the cytotoxic effector cells (comprising NK + K) to 6%. With the mouse mastocytoma cells (P815), which are not susceptible to NK, similar proportions of lymphocytes formed conjugates, and 6-9% were active as K cells. By an in situ rosetting assay a significant fraction of the TBC and cytotoxic effector cells bound either C3b or C3bi in both systems, with a certain predominance of C3bi-binding cells among the K cells. However, by indirect immunofluorescence, significantly more OKT3+ cells than OKM1+ cells were TBC or cytotoxic in the Chang cell system, whereas the OKT3+/OKM1+ ratios for both TBC and cytotoxic cells were 1:1 in the mouse mastocytoma system. The results indicate that TBC, NK and K cells are heterogeneous with respect to surface marker expression and that effector cells of different phenotypes predominate in different target systems.     

Identification of macromolecular insoluble cold globulin (MICG) as a new marker shared by NK and ADCC cells, but not expressed by NC cells.

We report here that cytotoxic pretreatment of spleen cells from six different strains of young adult mice with a monospecific rabbit antiserum against macromolecular insoluble cold globulin (MICG) effectively abrogates spontaneous NK activity directed towards YAC-1 tumour cells. MICG is a 225,000 molecular weight glycoprotein that is present in the plasma membrane of adult thymocytes and peripheral T cells, as well as in embryonic prothymocytes, but absent in granulocytes and B lymphocytes. The diminished NK activity in lymphocyte populations selectively depleted of MICG+ cells could not be restored by in vitro exposure to the NK boosting agents interleukin-2 (IL-2) and interferon. Lymphokine-activated spleen NK cells, generated by 48 hr preculture with IL-2 or interferon, expressed high levels of MICG surface antigen, moderate amounts of Thy 1.2 and, in striking contrast to spontaneous NK, very low to negligible amounts of AsGM1. Likewise, spontaneous NK cells in bone marrow were also shown to be both MICG+ and AsGM1+, while lymphokine-activated bone marrow NK cells remained MICG+, but lacked AsGM1. Thus, a clear distinction could be observed between spontaneous and activated NK cells with respect to differential expression of MICG and AsGM1. MICG was also detected on ADCC effector cells, whereas no surface MICG could be found on NC cells. These data are in line with the view that at least certain types of NK cells develop along a common lineage with T lymphocytes in the mouse.     

Lymphokine-activated killer cells and aging in mice: significance for defining the precursor cell

The present study was undertaken to define the cell populations which mediate lymphokine-activated killer (LAK) cell activity in mice. Because old mice exhibit markedly decreased to nondetectable natural killer (NK) cell activity, this age-associated change provided an advantageous system to examine the contribution of NK and T cells to LAK activity. Spleen cells from either young (6-9 weeks) or old (20-26 months) mice were cultured with 1000 units/ml of recombinant interleukin 2 (rIL 2) for 3-5 days. The cells were then tested in a 51 Cr-release assay for their cytotoxicity against NK-resistant fresh tumor cells (MCA-102). The LAK activity exhibited by spleen cells from old mice following 5 days of culture was equivalent to that developed by spleen cells of young mice. This result was contrary to what would be anticipated if mature NK cells comprise the primary precursors of LAK activity, and required further elucidation. The Thy-1 and asialo GM1 (ASGM1) phenotypes of LAK precursor and effector cells were therefore examined by depletion techniques using the appropriate antibodies plus complement. The results using spleen cells harvested after 5 days of culture with rIL 2 showed that LAK effector cells which developed from spleen cells of both young and old mice were predominantly Thy-1+ (85.3% young; 91.8% old) and some coexpressed ASGM1. Spleen cells were treated prior to culture to study the precursor cells. Development of LAK activity by spleen cells from both young and old mice was greatly reduced by pretreatment with anti-ASGM1 plus complement. However, since spleen cells of old mice exhibit very low mature NK activity, these data suggest that the LAK precursors, at least in old mice, may be ASGM1+ NK precursor cells rather than mature ASGM1+ NK effector cells. In addition, treatment with anti-Thy-1 plus complement inhibited generation of a significant proportion of LAK activity only in the spleens of old mice, suggesting a qualitative difference in LAK precursor cells with age and supporting the heterogeneity of the cells which are capable of developing LAK activity.     

Binding of Leukaemic Blasts to Various Subpopulations of Lymphokine-Activated Killer Cells

Conjugate formation by natural killer (NK)-resistant and N K-sensitive leukaemic cell lines with fresh and IL-2-stimulated (lymphokine-activated killer, LAK) peripheral blood lymphocytes (PBL) was studied by a flow cytofluorometry method with double staining, A significant difference in binding of NK-resistant T-cell lymphoma (HuT 78) and NK-sensitive myeloid (K562) blasts to fresh PBL was observed ( P <0.01). Activation of lymphocytes with IL-2 resulted in a significant increase of binding and killing of both K562 and HuT 78. However, in the case of blasts from NK-resistant cell line Daudi a similar conjugate formation with fresh PBL and LAK effectors was observed, despite a significant increase in killing. Various subpopulalions of LAK effectors (CD3, CD16 and CD56 positive) displayed similar binding activity towards myeloid (K562) and lymphoid (Raji) blasts, which shows that conjugate formation occurs not only with NK-cell-derived. but also with T-cell-derived LAK cells. The blocking of CD71 antigen (transferrin receptor) on K562 blasts inhibited binding of cytotoxic lymphocytes, which was mostly due to the blocking of binding of CD56⁺ subpopulation.
Our results indicate that the resistance of leukaemic blasts to cell-mediated cytotoxicity may depend on different (and probably several) steps of this process.     

Recombinant interleukin-2 inhibits growth of human tumor xenografts in congenitally athymic mice

Administration of human recombinant interleukin-2 (RIL-2) into congenitally athymic (nu/nu) mice carrying subcutaneous transplants of HeLa, HU 609T and T24B human carcinoma cells partially inhibited growth of the human tumor xenografts. In vitro activation of nu/nu spleen cells with human RIL-2 resulted in generation of killer cells showing in the 51Cr cytotoxicity assay similar levels of cytolysis as RIL-2-activated spleen cells from heterozygous (nu/+) mice. The RIL-2-activated (LAK) cells were cytotoxic for a variety of mouse and human tumors, reaching the peak of their cytotoxic activity after 3 days of cultivation in the RIL-2-containing medium. The cytotoxic activity of activated nu/nu spleen cells was significantly reduced by treatment with antibody against glycolipid asialo GM1, the differentiation antigen of natural killer (NK) cells. This finding suggests that in addition to the conventional, asialo GM1- LAK cells, asialo GM1+ activated NK cells participated in the cytotoxicity displayed by the IL-2-activated nu/nu killer spleen cells.